The present invention is directed to inhibitors of selectin mediated inflammation and is particularly directed to inhibitors derived from PSGL-1, the ligand for P-selectin, and novel oligosaccharides derived from PSGL-1.
The United States government has rights in this invention by virtue of National Institutes of Health Grant PO1 HL54804 and RO1 HL 43510 to Richard D. Cummings and Rodger P. McEver.
The selectins are a family of three Ca.sup.2+ -dependent membrane-bound lectins that initiate adhesion of leukocytes to platelets or endothelial cells under the shear forces found in the venular circulation (Laskey, (1992) Science 258, 964-969; Bevilacqua, et al., (1993) J. Clin. Invest. 91, 379-387; McEver (1994) Curr. Opin. Immunol. 6, 75-84). L-selectin, expressed on leukocytes, binds to constitutive or inducible ligands on endothelial cells. E-selectin, expressed by cytokine-activated endothelial cells, and P-selectin, expressed by thrombin-activated platelets and endothelial cells, bind to ligands on myeloid cells and subsets of lymphocytes.
P-selectin is a calcium-dependent carbohydrate-binding protein that is expressed on the surfaces of activated platelets and endothelium in response to thrombin and other agonists (McEver, et al. (1995) J. Biol. Chem., 270, 11025-11028; Varki, A. (1994) Proc. Natl. Acad. Sci., U.S.A. 91, 7390-7397; Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-872). Through its binding to glycoconjugate-based counterreceptors on leukocytes, P-selectin mediates rolling adhesion of these cells on activated platelets and endothelium (Lawrence and Springer (1991) Cell 65, 852-873; Moore, et al. (1995) J. Cell. Biol. 128, 661-671). Both sialic acid and fucose are components of the P-selectin counterreceptors on leukocytes (Corral, et al. (1990) Biochem. Biophys. Res. Comm. 172, 1349-1356; Moore, et al. (1992) J. Cell. Biol. 118, 445-456; Sako, et al. (1993) Cell 75, 1179-1186).
Although the selectins interact weakly with small sialylated, fucosylated oligosaccharides such as sialyl Lewis x (sLe.sup.x ; NeuAc.alpha.2-3Gal.beta.1-4[Fuc.alpha.1-3]GlcNAc) (Foxall, et al., (1992) J. Cell Biol. 117, 895-902; Varki, (1992) Curr. Opin. Cell Biol. 257, 257-266), they bind with higher affinity to glycans displayed on a limited number of glycoproteins (Moore, et al., (1992) J. Cell Biol. 118, 445-456; Lasky, et al., (1992) Cell 69, 927-938; Levinovitz, et al. (1993) J. Cell Biol. 121, 449-459; Walcheck, et al., (1993) J. Exp. Med. 178, 853-863; Baumhueter, et al., (1993) Science 262, 436-438; Berg, et al., (1993) Nature 366, 695-698) or proteoglycans (Norgard-Sumnicht, et al., (1993) Science 261, 480-483). Oligosaccharides containing sialyl Lewis x (sLe.sup.x), NeuAc.alpha.2-3Gal.beta.1-4[Fuc.alpha.1-3]GlcNAc.beta.1-R, a determinant present on leukocyte surfaces, inhibit adhesion of leukocytes to P-selectin (Polley, et al., (1991) Proc. Natl. Acad. Sci. USA 88, 6224-6228; Foxall, et al. (1992) J. Cell Biol. 117, 895-902). However, expression of slex on cell surfaces is not sufficient for high affinity binding of cells to P-selectin, since non-myeloid cells that express high levels of sLe.sup.x bind poorly to P-selectin compared to myeloid cells (Zhou, et al. (1991) J. Cell Biol. 115, 557-564). The high affinity ligands are potentially important as physiologic mediators of selectin-mediated leukocyte adhesion during inflammation. Thus, understanding the structural basis for high affinity recognition of these glycoconjugates by the selectins has attracted increasing interest.
A subset of the high affinity selectin ligands consists of mucin-like glycoproteins (McEver, et al. (1995)). One sialomucin ligand for P-selectin is expressed by human neutrophils and the human promyelocytic HL-60 cell line (Moore, et al., (1992); (Moore, et al., (1994) J. Biol. Chem. 269, 23318-23327). Leukocytes express a single high affinity ligand for P-selectin, termed P-selectin glycoprotein ligand-1 (PSGL-1) (Moore, et al., (1995); Moore, et al., (1992); Sako, et al., (1993); Norgaard, et al. (1993) J. Biol. Chem. 268, 12764-127748; Moore, et al. (1994) J. Biol. Chem. 269, 23318-23327). Binding of P-selectin to the ligand is Ca.sup.2+ dependent and is abolished by treatment of the ligand with sialidase.
PSGL is a homodimer with two disulfide-linked subunits with relative molecular masses of approximately 120,000 as assessed by SDS-PAGE (Moore, et al., 1992). Each subunit has no more than three N-glycans, but has many clustered, sialylated O-glycans (Moore, et al. (1992); Norgard, et al. (1993) J. Biol. Chem. 268, 12764-12774). The extracellular domain of PSGL-1 is highly extended, a characteristic feature of mucin-like proteins. PSGL-1 is a type 1 membrane protein with an extracellular domain containing many serines, threonines, and prolines, including a series of decameric repeats (15 in human promyelocytic HL-60 cells and 16 in human leukocytes) (Sako, et al. (1993) Cell 75, 1179-1186; Moore, et al. (1995) J. Cell Biol. 128, 661-671; Veldman, et al. (1995) J. Biol. Chem. 270, 16470-16475). Following an 18-residue signal peptide, there is a propeptide spanning residues 19-41 that is removed from PSGL-1 following its synthesis in leukocytes (Sako, et al. (1993); Vachino, et al. (1995) J. Biol. Chem. 270, 21966-21974). The extracellular domain of the processed mature protein extends from residues 42 to 318, and is followed by a 25-residue transmembrane domain at a 69-residue cytoplasmic tail. It carries many unmodified sialic residues as well as the sLe.sup.x antigen. The ligand contains at least one PNGaseF-sensitive N-linked glycan that P-selectin does not require for recognition. In contrast, it has clustered, sialylated, O-linked oligosaccharides that render the polypeptide backbone sensitive to cleavage by the enzyme O-sialoglycoprotease. Treatment of intact HL-60 cells with this enzyme eliminates the high affinity binding sites of P-selectin (Ushiyama, et al., (1993) J. Biol. Chem. 268, 15229-15237) and prevents cell adhesion to immobilized P-selection (Norgard, et al., (1993) J. Biol. Chem. 268, 12764-12774; Steininger, et al., Biochem. Biophys. Res. Commun. (1992) 188, 760-766), without affecting the overall surface expression of sLe.sup.x. These data suggest that this sialomucin, which carries only a small portion of the cell surface sLe.sup.x, corresponds to functionally important, high affinity binding sites for P-selectin on human myeloid cells.
Sako et al. (1993) isolated a cDNA derived from human HL-60 cells that encodes PSGL-1. The cDNA-derived sequence for each subunit of PSGL1 predicts a type 1 transmembrane protein of 402 amino acids. The extracellular domain has an N-terminal signal peptide from residues 1 to 18 and a putative propeptide from residues 19-41. Assuming cleavage of the ipropeptide, the extracellular domain of the mature protein begins at residue 42 and extends to residue 308. The sequence concludes with a 25-residue transmembrane domain and a 69 residue cytoplasmic tail. The extracellular domain is rich in serines and threonines that are potential sites of O-glycosylation. The extracellular domain contains three potential sites for addition of N-linked oligosaccharides as well as a single cysteine that might promote dimerization and three potential tyrosine sulfation sites at residues 46, 48 and 51.
Although previous studies have shown that sialylation and fucosylation of PSGL-1 are required for its binding to P-selectin, other post-translational modifications of PSGL-1 may also be important. PSGL-1 is highly O-glycosylated and contains sialylated and fucosylated O-linked poly-N-acetyllactosamine, including some glycans that terminate in sLe.sup.x (Moore, et al. (1994) J. Biol. Chem. 269, 23318-23327). sLe.sup.x or related glycans are not sufficient for high affinity binding of PSGL-1 to P-selectin. For example, sulfated compounds lacking either sialic acid or fucose can inhibit adhesion of leukocytes to P-selectin (Norgaard-Sumnicht, et al. (1993) Science 261, 480-483; Nelson, et al. (1993) Blood 82, 3253-3258; Cecconi, et al. (1994) J. Biol. Chem. 269, 15060-15066; Skinner, et al. (1989) Biochem. Biophys. Res. Comm. 164, 1373-1379). These data suggest that sulfation of PSGL-1 may be required for its high affinity binding to P-selectin. Recombinant PSGL-1 expressed in COS cells with a fucosyltransferase interacts with P-selectin in cell adhesion experiments (Sako, et al., 1993). However, the degree to which the oligosaccharides on recombinant PSGL-1 resemble those on the native glycoprotein ligand is unknown.
Although carbohydrates on PSGL-1 are critical for binding to selectins, no detailed chemical structures of the glycans are available. Much of the information about the glycosylation of the molecule has been obtained by enzymatic treatments of the native ligand and by studies on recombinant forms of PSGL-1 expressed in various cell types. While these indirect methods can provide valuable information about critical determinants on the ligand, detailed structural information on O-glycans from native PSGL-1 is essential to identify glycans that are important for ligand function and to provide a clearer understanding of why PSGL-1 is a ligand for P-and E-selectin, whereas other mucins such as CD43 are not.
It is therefore an object of the present invention to provide a method for making PSGL-1 which is normally glycosylated and sulfated.
It is another object of the present invention to provide a method and reagents for inhibiting binding of PSGL-1 to P-selectin and to other selectins.
It is a further object of the present invention to provide novel O-glycans, which are useful for modifying binding to P-selectin.